- Email: [email protected]
β
Journal Pre-proof Nuclear import of NLS- RARα is mediated by importin α/β
Jiao Ye, Liang Zhong, Ling Xiong, Jian Li, Lihua Yu, Wenran Dan, Zhen Yuan, Juanjuan Yao, Pengqiang Zhong, Junmei Liu, Dongdong Liu, Beizhong Liu PII:
S0898-6568(20)30044-9
DOI:
https://doi.org/10.1016/j.cellsig.2020.109567
Reference:
CLS 109567
To appear in:
Cellular Signalling
Received date:
16 October 2019
Revised date:
1 February 2020
Accepted date:
5 February 2020
Please cite this article as: J. Ye, L. Zhong, L. Xiong, et al., Nuclear import of NLSRARα is mediated by importin α/β, Cellular Signalling(2019), https://doi.org/10.1016/ j.cellsig.2020.109567
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© 2019 Published by Elsevier.
Journal Pre-proof Nuclear import of NLS- RARα is mediated by importin α/β Jiao Yea,b, Liang Zhongb, Ling Xionga, Jian Lib, Lihua Yua, Wenran Dana, Zhen Yuanb, Juanjuan Yaoa, Pengqiang Zhonga, Junmei Liua, Dongdong Liub, Beizhong Liua,b*
a
Central Laboratory of Yong-Chuan Hospital, Chongqing Medical University,
Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education,
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b
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Chongqing 402160, China
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Department of Laboratory Medicine, Chongqing Medical University, Chongqing
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400016, China
Correspondence
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Beizhong Liu, Central Laboratory of Yong-Chuan Hospital, Chongqing Medical
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University,
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Chongqing 402160, China.
Email: [email protected]
Abstract
Fax number: 0086-023-85368122
Journal Pre-proof The promyelocytic leukemia-retinoic acid receptor α (PML/RARα) is hypothesized to play a vital role in the pathogenesis of acute promyelocytic leukemia (APL). A previous study has demonstrated that PML/RARα is cleaved by neutrophil elastase (NE) in early myeloid cells, which leads to an increase in the nuclear localization signal (NLS) in RARα and in the incidence of APL. In this study, we explored the effects of NLS-RARα on acute myeloid leukemia (AML) cells and studied the
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mechanism of its localization. LV-NLS-RARα recombinant lentivirus and negative
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control LV-NC lentivirus were transfected into HL-60 cells and U937 cells while and all groups were treated
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mutant NLS-RARα were transfected into U937 cells,
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with 1α, 25-dihydroxyvitamin D3(1,25D3). The results showed that NLS-RARα was located mainly in the nucleus while mutant NLS-RARα was located in the cytoplasm.
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Overexpression of NLS-RARα downregulated the expression of CD11b, CD11c,
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CD14, and three forms of CEBPβ compared to the overexpression of NC and mutant
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NLS-RARα. It was speculated that the abnormal localization of NLS-RARα was mediated via importin- α/β in the pathogenesis of APL. By producing point mutations in the two NLSs in NLS-RARα, we showed that the nuclear import of NLS-RARα was mainly dependent on the NLS of the RARα portion. Subsequently, we found that importin-α1 (KPNA2)/importin-β1 (KPNB1) participates in the nuclear transport of NLS-RARα. Taken together, abnormal localization of NLS-RARα blocks the differentiation of APL cells, and nuclear localization of NLS-RARα depends on NLS of the RARα portion and is mediated via binding with importin-α/β.
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Keywords: Acute promyelocytic leukaemia; nuclear localisation signal; NLS-RARα; importin α/β; differentiation
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1. Introduction
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Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia
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(AML) characterized by a restriction of in differentiation and increased self- renewal
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of leukemic progenitor cells [1]. It manifests through a t(15;17) translocation, which
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fuses the retinoic acid receptor α (RARα) gene to the promyelocytic leukemia (PML) gene and results in a fusion protein, promyelocytic leukemia retinoic acid receptor α
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(PML/RARα) [2]. At present, the rate of remission of APL has improved by treatment
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with a combination of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO);
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however, 5-10% of the patients relapse or become resistant -5]. Until now, nearly all APL research has focused on the PML/RARα fusion protein. Lane and Ley demonstrated that PML/RARα could be cleaved by neutrophil elastase (NE), and NE-dependent PML/RARα cleavage activity exists in primary mouse and human APL cells [6]. Further, Lane and Ley showed that a high level of PML/RARα expression was associated with cellular toxicity that was dependent on the expression of NE [7]. In previous studies, many fusion proteins containing the RARα portion, rather than PML/RARα, have been linked to the pathogenesis of APL [8, 9]. The nuclear localization signal retinoic acid receptor α (NLS-RARα), which is a novel protein
Journal Pre-proof derived from the cleavage of PML/RARα by NE, contains an RARα portion and may play a significant role in APL. In previous studies, we found that NLS-RARα mainly localizes to the nucleus as compared to RARα, and inhibits the differentiation and promotes the proliferation of AML cells [10-13]. Many studies have demonstrated that the genesis and development of several cancers are associated with alterations in
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the nuclear-cytoplasmic transport of proteins [14-17]. Therefore, we speculated that
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the abnormal localization of NLS-RARα might be associated with the pathogenesis of
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APL. However, the mechanism underlying the control of NLS-RARα translocation
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between the cytoplasm and the nucleus is still unclear.
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Generally, most macromolecules enter the nucleus through nuclear pore complexes (NPCs) based on the NLS [18, 19]. Although there are several major nuclear transport
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pathways, classical nuclear import begins with recognition of the exposed lysine-rich
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NLS by the karyopherin α heterodimer, importin-α/β [20, 21]. Ivermectin is a specific
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importin-α/β inhibitor that does not affect other nuclear import pathways [22]. Importazole, a specific importin-β inhibitor, can also restrict this transport pathway by interrupting the binding of importin β to RanGTPase [23]. To date, the consensus NLS in PML has been identified and maps to the nuclear-targeting sequence 486
RKVIK 490 [24], whereas that of RARα is located in the DNA-binding domain
159
RNKKKK 164 [25]. Although the NLS-RARα protein contains two NLSs, one each
from PML and RARα, their precise roles in the localization of NLS-RARα are unknown. In the present study, we demonstrated that the abnormal nuclear accumulation of
Journal Pre-proof NLS-RARα could block differentiation of AML cells. Further, we confirmed that the localization of NLS-RARα mainly depended on the NLS from the RARα portion of NLS-RARα, and NLS- mutant NLS-RARα which was in the cytoplasm did not block differentiation of AML cells compared with it in the nucleus. Finally, we found that
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NLS-RARα entered the nucleus via binding to importin-α/β through the NLS.
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2. Material and Methods
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2.1. Cell culture
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NB4 (Chinese Academy Cell Bank, China), and the HL60 and U937 (Chinese
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Academy Cell Bank, China) leukemia cells were cultured in RPMI 1640 (Gibco Life
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Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, USA) containing penicillin (100 U/mL) and streptomycin (100 mg/mL) in an incubator with 5% CO 2 at 37℃. The U937 and HL60 cells were treated with 1α, 25-dihydroxyvitamin D3 (1,25D3, MCE, USA) at a final concentration of 400 nM. 293T cells are the most common mammalian cell type used for transient transfection; however, the transient transfection is inefficient in AML cells [26]. To explore the localization of recombinant mutated NLS-RARα, we used 293T cells. 293T cells (saved previously in our laboratory) were cultured in DMEM (Gibco Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum
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containing penicillin (100 U/mL) and streptomycin (100 mg/mL). All cells were incubated under 5% CO 2 at 37℃
2.2. Immunofluorescence staining
The 293T cells were seeded on coverslips in 24-well plates and cultured overnight at
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37 ℃. They were washed with phosphate-buffered saline (PBS) three times. AML
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cells were also washed with ice-cold PBS three times. Cells growing on coverslips in
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the 24-well plates were fixed in 4% paraformaldehyde for 20 min and permeabilized
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with 0.1% Triton X-100 in PBS for 22 min. Goat serum was added to the plates, and the cells were blocked at room temperature for 1 h. mouse HA tag antibody (1:200;
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Abcam, UK), rabbit KPNA2 antibody (1:200; Abcam, UK), or rabbit KPNB1
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antibody (1:200; Abcam, UK) was added to the plates and incubated at 4˚C overnight.
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The cells were then incubated with rabbit Alexa™ Fluor 488 (1:200; Abcam, UK) , mouse TRITC-conjugated secondary antibody (1:200; Beijing Zhongshan Golden Bridge Biotechnology, China) diluted in 0.1% Triton X-100. Subsequently, the nuclei were stained with DAPI by incubating for 10 min at room temperature. The cells were rinsed with PBS after each step.
2.3. Quantitative real-time polymerase chain reaction (qRT-PCR)
The oligonucleotide primers (Sangon Biotech, China) used in this study are listed in
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Table 1. For qRT-PCR, total cellular RNA was isolated using TRIZOL reagent (Takara, Japan). The total RNA was then reverse-transcribed using the PrimeScript RT reagent kit (Takara, Japan). , Each PCR mix contained 3 μL of cDNA, 1.5 μL (10 μM) of each primer, and 15 μL TB Green Premix Ex Taq Ⅱ (Takara, Japan), and sterile water was added until a total volume of 30 μL was achieved. β-actin served as an internal
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positive control.
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2.4. Plasmid construction
Table
1.
A
eukaryotic
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The primers used for plasmid construction and PCR-based gene targeting are listed in expression
vector
with
the
fusion
protein,
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pcDNA3.1-FLAG-KPNA2 was constructed. The KPNA2 plasmid was verified by
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enzymatic double digestion. A point mutation in NLS-RARα was constructed using
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GeneCreate (GeneCreate Bio. Inc, China) Table 1. Sequences used for PCR and qRT-PCR
Primers CD11b-F CD11b-R CD11c-F CD11c-R CD14-F CD14-R CEBPβ-F CEBPβ-R NLS-RARα-F NLS-RARα-R β-Actin-F β-Actin-R KPNA4-F
Primer sequence (5′→3′) CGCCATCATCTTACGGAACC CTGCCTGAACATCGCTACC AGAGCTGTGATAAGCCAGTTCC AATTCCTCGAAAGTGAAGTGTGT AAGCACTTCCAGAGCCTGTC TCGTCCAGCTCACAAGGTTC TACTACGAGGCGGACTGCTTGG CCAGCGGCTCCAGGTACGG ACCTCCAAGGCAGTCTC CCCCATAGTGGTAGCCT TGACGTGGACATCCGCAAAG CTGGAAGGTGGACAGCGAGG CTGTGTACGAGAGCGTGGTT
Journal Pre-proof KPNA4-R KPNA2-F KPNA2-R KPNA2-F※ KPNA2-R※
TATCAGCCCCCTGAAGGTGA TGACCAGATGCTGAAGAGGA GCTGATTTTCCACATTGCTG CGCGGATCCATGTCCACCAACGAGAATGCTAATAC CCGGAATTCCTACTTATCGTCGTCATCCTTGTAATCAAAGTTAA AGGTCCCAGGAGCCCCATCCTGAACTTGGA
Note: F stands for forward; R stands for reverse. ※Primers used for amplification of total sequence gene
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2.5. Transfection and infection
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Totally 10 μg of plasmid and 10 μL of Neofect DNA transfection reagent (Neofect
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Biotech Co. Ltd, China) were each diluted in 250 μL DMEM. After mixing gently and
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incubating for 20 min at room temperature, the mixture was placed in 10 mm2 Petri dishes containing 9.5 mL of complete medium.
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Overexpression of mutant NLS-RARα in U937 and HL60 cells was achieved using a
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lentiviral vector. The vector was designed and synthesized by GenePharma
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(GenePharma Shanghai GenePharma Co., Ltd, China). The cells (5 × 104 ) were transfected with green fluorescent protein (GFP)-expressing lentiviral vector, LV5-NC, and mutant NLS-RARα-expressing recombinant lentivirus, LV5- mutant NLS-RARα, and 5 µg/mL polybrene (GenePharma, China) were added. The medium was refreshed after culturing for 24 h. Following 48-72 h of incubation, fluorescence was detected by using a fluorescence microscope. Subsequently, the cells were treated with 5 μg/mL puromycin (Abcam, UK) for 15 d and selected to generate stable cell lines.
2.6. Immunoprecipitation (IP)
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Magnetic beads were incubated with mouse HA antibody and mouse IgG for 2 h at room temperature. Subsequently, the cell lysates were immunoprecipitated with the beads by incubating overnight at 4℃ to detect the interacting proteins. The precipitates were separated by sodium dodecyl sulfate-polyacrylamide gel
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electrophoresis (SDS-PAGE) and analyzed by immunoblotting. Gels were detected by
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mass spectrometry. Mass spectrum results of IP were generated by GeneCreate
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(GeneCreate Bio. Inc, China).
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2.7. Western blot analysis
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The cells in each group were washed with ice-cold PBS three times and lysed in RIPA
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buffer containing a protease inhibitor cocktail (Bimake, China). The cytoplasmic and
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nuclear fractions of the cultured cells were separated by using a nuclear and cytoplasmic protein extraction kit (Beyotime, China). Protein concentration was measured using the BCA protein assay kit. Fifty micrograms of total protein was separated by 10% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). The membranes were then blocked in 5% skim milk for 2 h at room temperature. The membranes were incubated with the primary antibody at 4˚C overnight. The following primary antibodies were used: mouse β-actin monoclonal antibody (1:1000; Boster, China); mouse α-tubulin monoclonal antibody (1:1000; Proteintech, USA); mouse HA-tag monoclonal
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antibody (1:1000; Genetex, USA); rabbit monoclonal antibodies to histone, CD11b, CD14, CD11c, and KPNB1 (1:1000; Abcam, UK); mouse CEBPβ monoclonal antibody (1:1000; Abcam, UK); and rabbit KPNA2 recombinant antibody (1:1000; Abcam, UK; Bimake, China). Then the membranes were incubated with goat anti-rabbit or goat anti‑ mouse secondary antibodies (1:4000 dilution; Zhongshan
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GoldenBridge Biotechnology Co., Ltd. China) for 1 h at room temperature. After
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washing with Tris‑ buffered saline containing Tween-20 (TBST), the immunoreactive
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complexes were visualized using an enhanced chemiluminescence system (Bio-Rad
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Laboratories Inc., Hercules, CA, USA). β-actin was used as an internal positive
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control.
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2.8. Statistical analysis
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GraphPad Prism 5 software (GraphPad, La Jolla, CA, USA) was used for statistical analysis. Data are shown as mean ± SD. A student’s t-test was used for comparison between two groups, and an ANOVA followed by a Tukey comparison test was applied for comparison between at least three groups. A value of p < 0.05 was considered significant.
3. Results
Journal Pre-proof 3.1 NLS-RARα suppresses 1,25D3-induced differentiation of HL60 and U937 cells
To explore the role of NLS-RARα in hematopoietic cell diff erentiation, we transfected the AML cell lines, HL60 and U937, with NLS-RARα (NR) or negative control (NC). The NC and NR groups were then treated with 1,25D3 in a
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time-dependent manner. Wright staining showed changes in the morphology of the
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cells (Figure 1A and D). qRT-PCR analysis showed a significant decrease in the
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expression of CD11b, CD11C, CD14, and CEBPβ mRNA in the NR cells treated with
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1,25D3 compared with NC cells treated with 1,25D3 (Figure 1B and E). The protein
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levels of CD11b, CD11C, CD14, and CEBPβ-1 (also known as LAP*), CEBPβ-2 (also known as LAP) and CEBPβ-3 (also known as LIP) were also decreased in the
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NR cells treated with 1,25D3 compared with NC cells treated with 1,25D3 (Figure 1C
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and F). Similar results were reported by Chunlan Xiao, Hao Song, and Xiu-Xiu Hu
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[11, 12, 27], which suggest that NLS-RARα expression is negatively correlated with the differentiation of AML cells.
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Figure 1. Overexpression of NLS-RARα inhibits differentiation of HL60 and U937 cells following treatment with 1,25D3. Wright-Giemsa staining images show the changes in cell morphology in HL60 (A) and U937 (D). Original magnification: 40x, 100x. The protein and mRNA levels of CD11b, CD11c, CD14, CEBPβ-1, CEBPβ-2, and CEBPβ-3 in HL60 (B-C), and U937 (E-F) cells were examined by western
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blotting and qRT-PCR, respectively. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared with LV-NC.
3.2 NLS-RARα localizes mostly to the nucleus.
We have shown that NLS-RARα located in the nucleus while RARα located in the
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cytoplasm [10]. Here, NLS-RARα was mainly localized to the nucleus in 293T cells,
western
blotting
(Figure
2A-D).
Similar
results
were
found
by
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by
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and U937, HL60, and NB4 cells showed transfection of NLS-RARα, as demonstrated
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immunofluorescence assays (Figure 2E - H). However, western blotting revealed that the localization of RARα was mostly in the cytoplasm (Figure 2I). Thus, the abnormal
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play an essential role.
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localization of NLS-RARα protein to the pathological mechanism of NLS-RARα may
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Figure 2. NLS-RARα is mainly located in the nucleus. Nuclear and cytoplasmic
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protein fractions were collected from NB4 cells (A), U937 cells (B), 293T cells (C) and
HL60
cells
(D)
after
transfection,
as
demonstrated
by
western blotting. Localization of NLS-RARα was detected in NB4 cells (F), U937 cells (G), 293T cells (H), and HL60 cells (I) by immunofluorescence staining. Original magnification: 200 x.
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3.3 Localization of NLS-RARα is dependent upon the NLS of the RARα portion.
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NLS-RARα has two primary NLSs, which include one from the PML, and the
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other from the RARα (Figure 3A). To demonstrate the role of the NLS in localization of NLS-RARα, we mutated the two NLSs individually or simultaneously (Figure 3B).
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NLS-RARα mutated in the NLS derived from the PML portion was labeled mut-1;
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NLS-RARα mutated in the NLS derived from RARα portion was labeled mut-2;
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NLS-RARα mutated in the NLS from both PML and RARα portions was labeled mut-12. Western blotting showed that the localization of NLS-RARα in the nucleus decreased in mut-2 and mut-12 (Figure 3C). Identical results were obtained in the immunofluorescence assay (Figure 3D). These data indicate that the NLS of the RARα portion in NLS-RARα is associated with the localization of NLS-RARα.
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Figure 3. NLS leads to the nuclear transport of NLS-RARα. (A) Arrows indicate the
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position of NE cleavage within the PML portion of P ML/RARα, after V420 and V430. The approximate predicted sizes of the peptide fragments generated by these cleavage events are shown. NE cleaves PML/RARα into PML without the NLS, and RARα obtains the NLS from PML (NLS-RARα). (B) Amino acid sequences of the three mutants used in this study are indicated in the figure. Mutated NLS of PML portion was named mut-1; Mutated NLS of RARα portion was named mut-2; Mutated NLSs of both PML and RARα portions were named mut-12. (C) Western blot assays determined the cellular localization of the various NLS-RARα mutant protein. (D) Detection of the cellular localization of the NLS‑ RARα mutant proteins by
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immunofluorescence assay. Original magnification: 200 x. Ns : no significance, *p < 0.05, **p < 0.01.
3.4 Suppressing the nuclear localization of NLS-RARα promotes 1,25D3- induced differentiation of U937 cells.
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To examine whether the abnormal localization of NLS-RARα has a significant
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role in the pathogenesis of APL, we compared the condition of the cells when
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NLS-RARα localized to the nucleus with when NLS-RARα localized to the
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cytoplasm. We transfected U937 cells with the mut-2 lentivirus (Figure 4A) and used western blotting to detect the distribution of mut-2 in the cells. The result showed that
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mut-2 localized to the cytoplasm (Figure 4B), which was in accord with that in 293T
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cells. Subsequently, NR and mut-2 groups were treated with 1,25D3 in a
expression
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time-dependent manner. qRT-PCR analysis showed a significant decrease in the of
CD11b,
NLS-RARα-transfected
CD11C,
cells
treated
CD14, with
and 1,25D3
CEBPβ
mRNA
compared
with
in
the
mutant
NLS-RARα-transfected cells treated with 1,25D3 (Figure 4C). Similar results were observed by western blotting (Figure 4D). These findings collectively demonstrate that the abnormal localization of NLS-RARα has a significant role in APL.
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Figure 4. NLS‑ RARα inhibits the differentiation of U937 following treatment with 1,25D3 compared with mut-2 NLS‑ RARα. (A) Mut-2 NLS‑ RARα was transfected
Journal Pre-proof successfully in the U937 cells. (B) Mut-2 NLS‑ RARα mainly localized in the cytoplasm, as detected by western blotting. (C-D) The protein and mRNA levels of CD11b, CD11c, CD14, CEBPβ-1, CEBPβ-2, and CEBPβ-3 in the U937 cells were detected by western blotting and qRT-PCR. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as compared with LV-NLS‑ RARα.
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3.5 Localization of NLS-RARα is linked to KPNA2/KPNB1
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To determine the mechanism used by NLS-RARα to enter the nucleus, we
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performed an immunoprecipitation assay followed by a mass spectrometric analysis (Supplementary Table 1). We found that KPNA2 and KPNB1 interacted with
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NLS-RARα. KPNA2 is overexpressed in most cancers. We compared the expression
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of KPNA2 and KPNA4 in AML and normal cells and found that KPNA2 was
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overexpressed in AML cells (Figure 5A). To demonstrate that KPNA2/KPNB1 interacts with NLS-RARα, we first constructed the pcDNA3.1-FLAG-KPNA2 plasmid, verified it by agarose gel electrophoresis (Figure 5B), and then transfected it into 293T cells. The interaction between KPNA2 and NLS-RARα was analyzed by immunoprecipitation and immunofluorescence analyses. As shown in Figure 5C, the co-localization of NLS-RARα and KPNA2/KPNB1 was detected in the nucleus. Further, the co-immunoprecipitation assay demonstrated that KPNA2 interacts with NLS-RARα (Figure 5D). Thus, KPNA2/KPNB1 may play a crucial role in the localization of NLS-RARα.
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Figure 5. NLS‑ RARα enters the nucleus via binding with importin α/β (A) KPNA2 was overexpressed in AML cells. (B) Construction of Flag-KPNA2 plasmids is successful. Lanes 6, 12: Vector; Lanes 2-5: Double digestion using BamHI and EcoRI; lanes 8-11: integral plasmids of Flag-KPNA2. The red arrow indicates NLS‑ RARα bands. The yellow arrow indicates vector bands. Marker 1: 1000 bp; Marker 2: 5000 bp; Marker 3: 10,000 bp. (C) Immunofluorescence analysis of KPNA2 (green) and
Journal Pre-proof KPNB1 (green) with NLS‑ RARα (red) localization in 293T cells. Original magnification: 200 x. (D)Interaction between KPNA2 and NLS‑ RARα in 293T cells was detected by co- immunoprecipitation. (E) Schematic diagram depicting the mechanism of NLS‑ RARα localization in APL cells.
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3.6 Specific inhibitors of the importin-α/β-dependent nuclear transport, ivermectin
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and importazole, decreased nuclear localization of NLS-RARα.
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To further demonstrate that the nuclear transport of NLS-RARα depends on
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importin-α/β, we used two specific inhibitors of importin-α/β, ivermectin and importazole. 293T cells transfected with NLS-RARα were treated with importazole at
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concentrations of 0, 10, 20, and 30 µM for 24 h. Western blotting and a decrease in
nuclear localization of
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immunofluorescence assays showed
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NLS-RARα(Figure 6A and B). Similarly, 293T cells transfected with NLS-RARα were treated with ivermectin at concentrations of 0, 5, 10, and 15 µM for 24 h. Reduction in the nuclear localization of NLS-RARα was observed by western blotting and immunofluorescence assays (Figure 6C and D). Similar results were observed in HL60 NR (Figure 6E) and NB4 NR cells (Figure 6F) that were treated with importazole at concentrations of 0, 5, 10, and 15 µM and in HL60 NR cells (Figure 6G) and U937 NR cells (Figure 6H) treated with ivermectin at co ncentrations of 0, 4, 8, and 12. These results confirm that the importin-α/β pathway regulates the localization
of
NLS-RARα
in
the
nucleus.
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Figure 6. Nuclear localization of NLS‑ RARα is inhibited by ivermectin and importazole. (A-D) Western blotting and immunofluorescence analysis shows a dose-dependent decrease in NLS‑ RARαin the nucleus of 293T cells. Original
Journal Pre-proof magnification: 200 x. (E-H) NLS‑ RARα decreased in the nucleus of AML cells treated with ivermectin and importazole.
4. Discussion
The PML/RARα protein, a hallmark of APL, is associated with the outcome of APL
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treatment [28, 29]. However, according to some recent studies, PML/RARα is not
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necessary for the development of APL [6, 7]. In early myeloid cells, the PML/RARα
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fusion protein can be cleaved by NE, generating the NLS- negative PML and
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NLS-RARα. Both this paper and previous studies have shown that NLS-RARα can block the differentiation of AML cells [12, 13] (Figure 1A-E). T These results suggest
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that NLS-RARα plays a role in the pathogenic mechanism of APL. Further, we found
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that NLS-RARα mainly localized to the nucleus unlike RARα [10](Figure 2A-H). In
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previous studies, yeast two-hybrid assay results have shown that NLS-RARα could be associated with ubiquilin 1(UBQLN1) and JTV1 [30, 31], implying that the abnormal localization of NLS-RARα results in protein-protein interactions. Such interactions may change the original signaling pathway and accelerate the development of APL. Furthermore, the abnormal localization of the proteins is involved in many cancers [14, 32]. We postulate that the localization mechanism of NLS-RARα plays an important role in APL. Based on the predicted NLS and previous studies, NLS-RARα includes two NLSs, one each from PML and RARα (Supplementary Table 2). Previously, we speculated
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that the additional NLS increases the nuclear localization of NLS-RARα. In this study, we mutated the two NLSs and found that mutating only the NLS from the RARα portion could significantly decrease the nuclear localization of NLS-RARα. It demonstrated that the RARα portion plays a major role in NLS-RARα (Figure 3C-D). This is the first time that the NLS of the RARα portion have been shown to plays a major role in NLS-RARα localization. Further, the NLS-mutant NLS-RARα did not
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affect the differentiation of AML cells compared with wild NLS-RARα (Figure 4A-D).
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It verifies the nuclear accumulation of NLS-RARα restricted the differentiation once
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again.
The present study also uncovered the mechanism of NLS-RARα localization to the NLS-RARα possesses two
NLSs.
In
the
importin-α/β pathway,
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nucleus.
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cNLS-containing proteins assemble into a ternary complex with importin α and β [33].
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To determine whether NLS-RARα localization was mediated by importin α/β, immunoprecipitation and immunofluorescence analyses were performed. The results showed that NLS-RARα bound to importin-α1, which co- localized with importin-α/β (Figure 5C-D). Moreover, localization of NLS-RARα to the nucleus was markedly decreased in the presence of ivermectin and importazole, which specifically inhibit the importin-α/β pathway and importin-β pathway, respectively (Figure 6A-H). Taken together, these results showed that NLS-RARα is transported to the nucleus via the importin-α/β pathway.
RARα belongs to the nuclear receptor superfamily, which that recognizes retinoic
Journal Pre-proof acid (RA) as the ligand [34]. RARα regulates the transcription of genes containing the RAR response elements (RARE) [35]. In many types of cells, RARα is localized to the cytoplasm. In a previous study, RARα was localized to the cytoplasm under standard culture conditions, but in the presence of cytokines and RA, RARα gradually translocated to the nucleus [36]. However, the mechanism underlying the localization of RARα to the nucleus is still unclear. A study by Mezaki et al.
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demonstrated that endogenous RARα localized to the cytoplasm because the NLS of
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the endogenous RARα proteins was temporarily masked [25]. Further, a study by
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Huang showed that changes in the subcellular localization of RARα could be
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regulated by RA and protein kinase C [37]. Further, RARα is the phosphorylation substrate for the C-subunit of protein kinase A (PKA) and Ser-371 of RARα was
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predicted to be the phosphorylation site [38]. Studies have demonstrated that protein
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kinases regulate the localization of the target proteins containing NLS via the
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phosphorylation of protein kinase phosphorylation sites adjacent to the NLS [39, 40]. Hence, we speculated that RARα was normally located in the cytoplasm. However, the presence of ligands or phosphorylation, changed the conformation of RARα, resulting in exposure of the NLS, which allowed target proteins to enter the nucleus. NLS-RARα translocated to the nucleus based on the NLS of the RARα portion, and the transport of NLS-RARα may be analogous to that of RARα. Furthermore, we predicted that the putative phosphorylation sites were around the NLS in NLS-RARα using Netphos 3.1 Server (supplementary Figure 1). However, in APL patients, NLS-RARα localized to the nucleus. We speculate that NE cleaves PML/RARα,
Journal Pre-proof which results in conformational changes in NLS-RARα. This leads to exposure of the NLS and phosphorylation sites. However, further studies are needed to investigated this hypothesis. In conclusion, to the best of our knowledge, this is the first study to show the mechanism of nuclear localization of NLS-RARα, and to verify that nuclear accumulation of NLS-RARα decreases the differentiation of AML cells compared to
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its localization in the cytoplasm. These results suggest that NLS-RARα could be used
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as a biomarker in APL, and the transport pathway could be an alternative mechanism to
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modulate the pathogenic role of NLS-RARα in vitro.
Acknowledgments
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This work was funded by the National Natural Science Foundation of China (grant
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number 81772280) and the Chongqing Science and Technology Commission Fund
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（NO.cstc2018jscx-msybX0173）. Author contributions
Jiao Ye, Dongdong Liu and Juanjuan Yao conceived and designed the experiments; Jiao Ye, LiangZhong, Jiao Li, Li Xiong, Lihua Li, Pengqiang Zhong, performed the experiments; Zhen Yuan and Junmei Liu, analyzed the data; Jiao Ye wrote the paper. Declaration of competing Interest The authors declare that they have no conflict of interest. References [1]. de The H, Chen Z. Acute promyelocytic leukaemia: novel insights into the mechanisms of
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Declaration of competing Interest
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The authors declare that they have no conflict of interest.
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Highlights 1.the nuclear localization of NLS-RARα inhibits 1,25D3- induced differentiation of HL-60 and U937 cells. 2.NLS of RARα portion mediates the nuclear localization of NLS-RARα. 3. importin-α/β participates in the nuclear transport of NLS-RARα via binding to
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NLS.
Jiao Ye, Liang Zhong, Ling Xiong, Jian Li, Lihua Yu, Wenran Dan, Zhen Yuan, Juanjuan Yao, Pengqiang Zhong, Junmei Liu, Dongdong Liu, Beizhong Liu PII:
S0898-6568(20)30044-9
DOI:
https://doi.org/10.1016/j.cellsig.2020.109567
Reference:
CLS 109567
To appear in:
Cellular Signalling
Received date:
16 October 2019
Revised date:
1 February 2020
Accepted date:
5 February 2020
Please cite this article as: J. Ye, L. Zhong, L. Xiong, et al., Nuclear import of NLSRARα is mediated by importin α/β, Cellular Signalling(2019), https://doi.org/10.1016/ j.cellsig.2020.109567
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© 2019 Published by Elsevier.
Journal Pre-proof Nuclear import of NLS- RARα is mediated by importin α/β Jiao Yea,b, Liang Zhongb, Ling Xionga, Jian Lib, Lihua Yua, Wenran Dana, Zhen Yuanb, Juanjuan Yaoa, Pengqiang Zhonga, Junmei Liua, Dongdong Liub, Beizhong Liua,b*
a
Central Laboratory of Yong-Chuan Hospital, Chongqing Medical University,
Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education,
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b
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Chongqing 402160, China
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Department of Laboratory Medicine, Chongqing Medical University, Chongqing
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400016, China
Correspondence
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Beizhong Liu, Central Laboratory of Yong-Chuan Hospital, Chongqing Medical
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University,
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Chongqing 402160, China.
Email: [email protected]
Abstract
Fax number: 0086-023-85368122
Journal Pre-proof The promyelocytic leukemia-retinoic acid receptor α (PML/RARα) is hypothesized to play a vital role in the pathogenesis of acute promyelocytic leukemia (APL). A previous study has demonstrated that PML/RARα is cleaved by neutrophil elastase (NE) in early myeloid cells, which leads to an increase in the nuclear localization signal (NLS) in RARα and in the incidence of APL. In this study, we explored the effects of NLS-RARα on acute myeloid leukemia (AML) cells and studied the
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mechanism of its localization. LV-NLS-RARα recombinant lentivirus and negative
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control LV-NC lentivirus were transfected into HL-60 cells and U937 cells while and all groups were treated
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mutant NLS-RARα were transfected into U937 cells,
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with 1α, 25-dihydroxyvitamin D3(1,25D3). The results showed that NLS-RARα was located mainly in the nucleus while mutant NLS-RARα was located in the cytoplasm.
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Overexpression of NLS-RARα downregulated the expression of CD11b, CD11c,
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CD14, and three forms of CEBPβ compared to the overexpression of NC and mutant
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NLS-RARα. It was speculated that the abnormal localization of NLS-RARα was mediated via importin- α/β in the pathogenesis of APL. By producing point mutations in the two NLSs in NLS-RARα, we showed that the nuclear import of NLS-RARα was mainly dependent on the NLS of the RARα portion. Subsequently, we found that importin-α1 (KPNA2)/importin-β1 (KPNB1) participates in the nuclear transport of NLS-RARα. Taken together, abnormal localization of NLS-RARα blocks the differentiation of APL cells, and nuclear localization of NLS-RARα depends on NLS of the RARα portion and is mediated via binding with importin-α/β.
Journal Pre-proof
Keywords: Acute promyelocytic leukaemia; nuclear localisation signal; NLS-RARα; importin α/β; differentiation
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1. Introduction
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Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia
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(AML) characterized by a restriction of in differentiation and increased self- renewal
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of leukemic progenitor cells [1]. It manifests through a t(15;17) translocation, which
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fuses the retinoic acid receptor α (RARα) gene to the promyelocytic leukemia (PML) gene and results in a fusion protein, promyelocytic leukemia retinoic acid receptor α
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(PML/RARα) [2]. At present, the rate of remission of APL has improved by treatment
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with a combination of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO);
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however, 5-10% of the patients relapse or become resistant -5]. Until now, nearly all APL research has focused on the PML/RARα fusion protein. Lane and Ley demonstrated that PML/RARα could be cleaved by neutrophil elastase (NE), and NE-dependent PML/RARα cleavage activity exists in primary mouse and human APL cells [6]. Further, Lane and Ley showed that a high level of PML/RARα expression was associated with cellular toxicity that was dependent on the expression of NE [7]. In previous studies, many fusion proteins containing the RARα portion, rather than PML/RARα, have been linked to the pathogenesis of APL [8, 9]. The nuclear localization signal retinoic acid receptor α (NLS-RARα), which is a novel protein
Journal Pre-proof derived from the cleavage of PML/RARα by NE, contains an RARα portion and may play a significant role in APL. In previous studies, we found that NLS-RARα mainly localizes to the nucleus as compared to RARα, and inhibits the differentiation and promotes the proliferation of AML cells [10-13]. Many studies have demonstrated that the genesis and development of several cancers are associated with alterations in
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the nuclear-cytoplasmic transport of proteins [14-17]. Therefore, we speculated that
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the abnormal localization of NLS-RARα might be associated with the pathogenesis of
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APL. However, the mechanism underlying the control of NLS-RARα translocation
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between the cytoplasm and the nucleus is still unclear.
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Generally, most macromolecules enter the nucleus through nuclear pore complexes (NPCs) based on the NLS [18, 19]. Although there are several major nuclear transport
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pathways, classical nuclear import begins with recognition of the exposed lysine-rich
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NLS by the karyopherin α heterodimer, importin-α/β [20, 21]. Ivermectin is a specific
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importin-α/β inhibitor that does not affect other nuclear import pathways [22]. Importazole, a specific importin-β inhibitor, can also restrict this transport pathway by interrupting the binding of importin β to RanGTPase [23]. To date, the consensus NLS in PML has been identified and maps to the nuclear-targeting sequence 486
RKVIK 490 [24], whereas that of RARα is located in the DNA-binding domain
159
RNKKKK 164 [25]. Although the NLS-RARα protein contains two NLSs, one each
from PML and RARα, their precise roles in the localization of NLS-RARα are unknown. In the present study, we demonstrated that the abnormal nuclear accumulation of
Journal Pre-proof NLS-RARα could block differentiation of AML cells. Further, we confirmed that the localization of NLS-RARα mainly depended on the NLS from the RARα portion of NLS-RARα, and NLS- mutant NLS-RARα which was in the cytoplasm did not block differentiation of AML cells compared with it in the nucleus. Finally, we found that
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NLS-RARα entered the nucleus via binding to importin-α/β through the NLS.
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2. Material and Methods
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2.1. Cell culture
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NB4 (Chinese Academy Cell Bank, China), and the HL60 and U937 (Chinese
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Academy Cell Bank, China) leukemia cells were cultured in RPMI 1640 (Gibco Life
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Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, USA) containing penicillin (100 U/mL) and streptomycin (100 mg/mL) in an incubator with 5% CO 2 at 37℃. The U937 and HL60 cells were treated with 1α, 25-dihydroxyvitamin D3 (1,25D3, MCE, USA) at a final concentration of 400 nM. 293T cells are the most common mammalian cell type used for transient transfection; however, the transient transfection is inefficient in AML cells [26]. To explore the localization of recombinant mutated NLS-RARα, we used 293T cells. 293T cells (saved previously in our laboratory) were cultured in DMEM (Gibco Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum
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containing penicillin (100 U/mL) and streptomycin (100 mg/mL). All cells were incubated under 5% CO 2 at 37℃
2.2. Immunofluorescence staining
The 293T cells were seeded on coverslips in 24-well plates and cultured overnight at
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37 ℃. They were washed with phosphate-buffered saline (PBS) three times. AML
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cells were also washed with ice-cold PBS three times. Cells growing on coverslips in
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the 24-well plates were fixed in 4% paraformaldehyde for 20 min and permeabilized
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with 0.1% Triton X-100 in PBS for 22 min. Goat serum was added to the plates, and the cells were blocked at room temperature for 1 h. mouse HA tag antibody (1:200;
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Abcam, UK), rabbit KPNA2 antibody (1:200; Abcam, UK), or rabbit KPNB1
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antibody (1:200; Abcam, UK) was added to the plates and incubated at 4˚C overnight.
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The cells were then incubated with rabbit Alexa™ Fluor 488 (1:200; Abcam, UK) , mouse TRITC-conjugated secondary antibody (1:200; Beijing Zhongshan Golden Bridge Biotechnology, China) diluted in 0.1% Triton X-100. Subsequently, the nuclei were stained with DAPI by incubating for 10 min at room temperature. The cells were rinsed with PBS after each step.
2.3. Quantitative real-time polymerase chain reaction (qRT-PCR)
The oligonucleotide primers (Sangon Biotech, China) used in this study are listed in
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Table 1. For qRT-PCR, total cellular RNA was isolated using TRIZOL reagent (Takara, Japan). The total RNA was then reverse-transcribed using the PrimeScript RT reagent kit (Takara, Japan). , Each PCR mix contained 3 μL of cDNA, 1.5 μL (10 μM) of each primer, and 15 μL TB Green Premix Ex Taq Ⅱ (Takara, Japan), and sterile water was added until a total volume of 30 μL was achieved. β-actin served as an internal
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positive control.
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2.4. Plasmid construction
Table
1.
A
eukaryotic
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The primers used for plasmid construction and PCR-based gene targeting are listed in expression
vector
with
the
fusion
protein,
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pcDNA3.1-FLAG-KPNA2 was constructed. The KPNA2 plasmid was verified by
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enzymatic double digestion. A point mutation in NLS-RARα was constructed using
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GeneCreate (GeneCreate Bio. Inc, China) Table 1. Sequences used for PCR and qRT-PCR
Primers CD11b-F CD11b-R CD11c-F CD11c-R CD14-F CD14-R CEBPβ-F CEBPβ-R NLS-RARα-F NLS-RARα-R β-Actin-F β-Actin-R KPNA4-F
Primer sequence (5′→3′) CGCCATCATCTTACGGAACC CTGCCTGAACATCGCTACC AGAGCTGTGATAAGCCAGTTCC AATTCCTCGAAAGTGAAGTGTGT AAGCACTTCCAGAGCCTGTC TCGTCCAGCTCACAAGGTTC TACTACGAGGCGGACTGCTTGG CCAGCGGCTCCAGGTACGG ACCTCCAAGGCAGTCTC CCCCATAGTGGTAGCCT TGACGTGGACATCCGCAAAG CTGGAAGGTGGACAGCGAGG CTGTGTACGAGAGCGTGGTT
Journal Pre-proof KPNA4-R KPNA2-F KPNA2-R KPNA2-F※ KPNA2-R※
TATCAGCCCCCTGAAGGTGA TGACCAGATGCTGAAGAGGA GCTGATTTTCCACATTGCTG CGCGGATCCATGTCCACCAACGAGAATGCTAATAC CCGGAATTCCTACTTATCGTCGTCATCCTTGTAATCAAAGTTAA AGGTCCCAGGAGCCCCATCCTGAACTTGGA
Note: F stands for forward; R stands for reverse. ※Primers used for amplification of total sequence gene
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2.5. Transfection and infection
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Totally 10 μg of plasmid and 10 μL of Neofect DNA transfection reagent (Neofect
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Biotech Co. Ltd, China) were each diluted in 250 μL DMEM. After mixing gently and
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incubating for 20 min at room temperature, the mixture was placed in 10 mm2 Petri dishes containing 9.5 mL of complete medium.
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Overexpression of mutant NLS-RARα in U937 and HL60 cells was achieved using a
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lentiviral vector. The vector was designed and synthesized by GenePharma
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(GenePharma Shanghai GenePharma Co., Ltd, China). The cells (5 × 104 ) were transfected with green fluorescent protein (GFP)-expressing lentiviral vector, LV5-NC, and mutant NLS-RARα-expressing recombinant lentivirus, LV5- mutant NLS-RARα, and 5 µg/mL polybrene (GenePharma, China) were added. The medium was refreshed after culturing for 24 h. Following 48-72 h of incubation, fluorescence was detected by using a fluorescence microscope. Subsequently, the cells were treated with 5 μg/mL puromycin (Abcam, UK) for 15 d and selected to generate stable cell lines.
2.6. Immunoprecipitation (IP)
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Magnetic beads were incubated with mouse HA antibody and mouse IgG for 2 h at room temperature. Subsequently, the cell lysates were immunoprecipitated with the beads by incubating overnight at 4℃ to detect the interacting proteins. The precipitates were separated by sodium dodecyl sulfate-polyacrylamide gel
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electrophoresis (SDS-PAGE) and analyzed by immunoblotting. Gels were detected by
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mass spectrometry. Mass spectrum results of IP were generated by GeneCreate
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(GeneCreate Bio. Inc, China).
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2.7. Western blot analysis
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The cells in each group were washed with ice-cold PBS three times and lysed in RIPA
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buffer containing a protease inhibitor cocktail (Bimake, China). The cytoplasmic and
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nuclear fractions of the cultured cells were separated by using a nuclear and cytoplasmic protein extraction kit (Beyotime, China). Protein concentration was measured using the BCA protein assay kit. Fifty micrograms of total protein was separated by 10% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). The membranes were then blocked in 5% skim milk for 2 h at room temperature. The membranes were incubated with the primary antibody at 4˚C overnight. The following primary antibodies were used: mouse β-actin monoclonal antibody (1:1000; Boster, China); mouse α-tubulin monoclonal antibody (1:1000; Proteintech, USA); mouse HA-tag monoclonal
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antibody (1:1000; Genetex, USA); rabbit monoclonal antibodies to histone, CD11b, CD14, CD11c, and KPNB1 (1:1000; Abcam, UK); mouse CEBPβ monoclonal antibody (1:1000; Abcam, UK); and rabbit KPNA2 recombinant antibody (1:1000; Abcam, UK; Bimake, China). Then the membranes were incubated with goat anti-rabbit or goat anti‑ mouse secondary antibodies (1:4000 dilution; Zhongshan
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GoldenBridge Biotechnology Co., Ltd. China) for 1 h at room temperature. After
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washing with Tris‑ buffered saline containing Tween-20 (TBST), the immunoreactive
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complexes were visualized using an enhanced chemiluminescence system (Bio-Rad
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Laboratories Inc., Hercules, CA, USA). β-actin was used as an internal positive
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control.
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2.8. Statistical analysis
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GraphPad Prism 5 software (GraphPad, La Jolla, CA, USA) was used for statistical analysis. Data are shown as mean ± SD. A student’s t-test was used for comparison between two groups, and an ANOVA followed by a Tukey comparison test was applied for comparison between at least three groups. A value of p < 0.05 was considered significant.
3. Results
Journal Pre-proof 3.1 NLS-RARα suppresses 1,25D3-induced differentiation of HL60 and U937 cells
To explore the role of NLS-RARα in hematopoietic cell diff erentiation, we transfected the AML cell lines, HL60 and U937, with NLS-RARα (NR) or negative control (NC). The NC and NR groups were then treated with 1,25D3 in a
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time-dependent manner. Wright staining showed changes in the morphology of the
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cells (Figure 1A and D). qRT-PCR analysis showed a significant decrease in the
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expression of CD11b, CD11C, CD14, and CEBPβ mRNA in the NR cells treated with
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1,25D3 compared with NC cells treated with 1,25D3 (Figure 1B and E). The protein
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levels of CD11b, CD11C, CD14, and CEBPβ-1 (also known as LAP*), CEBPβ-2 (also known as LAP) and CEBPβ-3 (also known as LIP) were also decreased in the
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NR cells treated with 1,25D3 compared with NC cells treated with 1,25D3 (Figure 1C
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and F). Similar results were reported by Chunlan Xiao, Hao Song, and Xiu-Xiu Hu
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[11, 12, 27], which suggest that NLS-RARα expression is negatively correlated with the differentiation of AML cells.
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Figure 1. Overexpression of NLS-RARα inhibits differentiation of HL60 and U937 cells following treatment with 1,25D3. Wright-Giemsa staining images show the changes in cell morphology in HL60 (A) and U937 (D). Original magnification: 40x, 100x. The protein and mRNA levels of CD11b, CD11c, CD14, CEBPβ-1, CEBPβ-2, and CEBPβ-3 in HL60 (B-C), and U937 (E-F) cells were examined by western
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blotting and qRT-PCR, respectively. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared with LV-NC.
3.2 NLS-RARα localizes mostly to the nucleus.
We have shown that NLS-RARα located in the nucleus while RARα located in the
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cytoplasm [10]. Here, NLS-RARα was mainly localized to the nucleus in 293T cells,
western
blotting
(Figure
2A-D).
Similar
results
were
found
by
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by
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and U937, HL60, and NB4 cells showed transfection of NLS-RARα, as demonstrated
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immunofluorescence assays (Figure 2E - H). However, western blotting revealed that the localization of RARα was mostly in the cytoplasm (Figure 2I). Thus, the abnormal
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play an essential role.
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localization of NLS-RARα protein to the pathological mechanism of NLS-RARα may
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Figure 2. NLS-RARα is mainly located in the nucleus. Nuclear and cytoplasmic
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protein fractions were collected from NB4 cells (A), U937 cells (B), 293T cells (C) and
HL60
cells
(D)
after
transfection,
as
demonstrated
by
western blotting. Localization of NLS-RARα was detected in NB4 cells (F), U937 cells (G), 293T cells (H), and HL60 cells (I) by immunofluorescence staining. Original magnification: 200 x.
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3.3 Localization of NLS-RARα is dependent upon the NLS of the RARα portion.
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NLS-RARα has two primary NLSs, which include one from the PML, and the
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other from the RARα (Figure 3A). To demonstrate the role of the NLS in localization of NLS-RARα, we mutated the two NLSs individually or simultaneously (Figure 3B).
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NLS-RARα mutated in the NLS derived from the PML portion was labeled mut-1;
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NLS-RARα mutated in the NLS derived from RARα portion was labeled mut-2;
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NLS-RARα mutated in the NLS from both PML and RARα portions was labeled mut-12. Western blotting showed that the localization of NLS-RARα in the nucleus decreased in mut-2 and mut-12 (Figure 3C). Identical results were obtained in the immunofluorescence assay (Figure 3D). These data indicate that the NLS of the RARα portion in NLS-RARα is associated with the localization of NLS-RARα.
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Figure 3. NLS leads to the nuclear transport of NLS-RARα. (A) Arrows indicate the
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position of NE cleavage within the PML portion of P ML/RARα, after V420 and V430. The approximate predicted sizes of the peptide fragments generated by these cleavage events are shown. NE cleaves PML/RARα into PML without the NLS, and RARα obtains the NLS from PML (NLS-RARα). (B) Amino acid sequences of the three mutants used in this study are indicated in the figure. Mutated NLS of PML portion was named mut-1; Mutated NLS of RARα portion was named mut-2; Mutated NLSs of both PML and RARα portions were named mut-12. (C) Western blot assays determined the cellular localization of the various NLS-RARα mutant protein. (D) Detection of the cellular localization of the NLS‑ RARα mutant proteins by
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immunofluorescence assay. Original magnification: 200 x. Ns : no significance, *p < 0.05, **p < 0.01.
3.4 Suppressing the nuclear localization of NLS-RARα promotes 1,25D3- induced differentiation of U937 cells.
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To examine whether the abnormal localization of NLS-RARα has a significant
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role in the pathogenesis of APL, we compared the condition of the cells when
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NLS-RARα localized to the nucleus with when NLS-RARα localized to the
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cytoplasm. We transfected U937 cells with the mut-2 lentivirus (Figure 4A) and used western blotting to detect the distribution of mut-2 in the cells. The result showed that
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mut-2 localized to the cytoplasm (Figure 4B), which was in accord with that in 293T
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cells. Subsequently, NR and mut-2 groups were treated with 1,25D3 in a
expression
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time-dependent manner. qRT-PCR analysis showed a significant decrease in the of
CD11b,
NLS-RARα-transfected
CD11C,
cells
treated
CD14, with
and 1,25D3
CEBPβ
mRNA
compared
with
in
the
mutant
NLS-RARα-transfected cells treated with 1,25D3 (Figure 4C). Similar results were observed by western blotting (Figure 4D). These findings collectively demonstrate that the abnormal localization of NLS-RARα has a significant role in APL.
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Figure 4. NLS‑ RARα inhibits the differentiation of U937 following treatment with 1,25D3 compared with mut-2 NLS‑ RARα. (A) Mut-2 NLS‑ RARα was transfected
Journal Pre-proof successfully in the U937 cells. (B) Mut-2 NLS‑ RARα mainly localized in the cytoplasm, as detected by western blotting. (C-D) The protein and mRNA levels of CD11b, CD11c, CD14, CEBPβ-1, CEBPβ-2, and CEBPβ-3 in the U937 cells were detected by western blotting and qRT-PCR. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as compared with LV-NLS‑ RARα.
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3.5 Localization of NLS-RARα is linked to KPNA2/KPNB1
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To determine the mechanism used by NLS-RARα to enter the nucleus, we
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performed an immunoprecipitation assay followed by a mass spectrometric analysis (Supplementary Table 1). We found that KPNA2 and KPNB1 interacted with
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NLS-RARα. KPNA2 is overexpressed in most cancers. We compared the expression
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of KPNA2 and KPNA4 in AML and normal cells and found that KPNA2 was
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overexpressed in AML cells (Figure 5A). To demonstrate that KPNA2/KPNB1 interacts with NLS-RARα, we first constructed the pcDNA3.1-FLAG-KPNA2 plasmid, verified it by agarose gel electrophoresis (Figure 5B), and then transfected it into 293T cells. The interaction between KPNA2 and NLS-RARα was analyzed by immunoprecipitation and immunofluorescence analyses. As shown in Figure 5C, the co-localization of NLS-RARα and KPNA2/KPNB1 was detected in the nucleus. Further, the co-immunoprecipitation assay demonstrated that KPNA2 interacts with NLS-RARα (Figure 5D). Thus, KPNA2/KPNB1 may play a crucial role in the localization of NLS-RARα.
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Figure 5. NLS‑ RARα enters the nucleus via binding with importin α/β (A) KPNA2 was overexpressed in AML cells. (B) Construction of Flag-KPNA2 plasmids is successful. Lanes 6, 12: Vector; Lanes 2-5: Double digestion using BamHI and EcoRI; lanes 8-11: integral plasmids of Flag-KPNA2. The red arrow indicates NLS‑ RARα bands. The yellow arrow indicates vector bands. Marker 1: 1000 bp; Marker 2: 5000 bp; Marker 3: 10,000 bp. (C) Immunofluorescence analysis of KPNA2 (green) and
Journal Pre-proof KPNB1 (green) with NLS‑ RARα (red) localization in 293T cells. Original magnification: 200 x. (D)Interaction between KPNA2 and NLS‑ RARα in 293T cells was detected by co- immunoprecipitation. (E) Schematic diagram depicting the mechanism of NLS‑ RARα localization in APL cells.
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3.6 Specific inhibitors of the importin-α/β-dependent nuclear transport, ivermectin
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and importazole, decreased nuclear localization of NLS-RARα.
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To further demonstrate that the nuclear transport of NLS-RARα depends on
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importin-α/β, we used two specific inhibitors of importin-α/β, ivermectin and importazole. 293T cells transfected with NLS-RARα were treated with importazole at
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concentrations of 0, 10, 20, and 30 µM for 24 h. Western blotting and a decrease in
nuclear localization of
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immunofluorescence assays showed
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NLS-RARα(Figure 6A and B). Similarly, 293T cells transfected with NLS-RARα were treated with ivermectin at concentrations of 0, 5, 10, and 15 µM for 24 h. Reduction in the nuclear localization of NLS-RARα was observed by western blotting and immunofluorescence assays (Figure 6C and D). Similar results were observed in HL60 NR (Figure 6E) and NB4 NR cells (Figure 6F) that were treated with importazole at concentrations of 0, 5, 10, and 15 µM and in HL60 NR cells (Figure 6G) and U937 NR cells (Figure 6H) treated with ivermectin at co ncentrations of 0, 4, 8, and 12. These results confirm that the importin-α/β pathway regulates the localization
of
NLS-RARα
in
the
nucleus.
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Figure 6. Nuclear localization of NLS‑ RARα is inhibited by ivermectin and importazole. (A-D) Western blotting and immunofluorescence analysis shows a dose-dependent decrease in NLS‑ RARαin the nucleus of 293T cells. Original
Journal Pre-proof magnification: 200 x. (E-H) NLS‑ RARα decreased in the nucleus of AML cells treated with ivermectin and importazole.
4. Discussion
The PML/RARα protein, a hallmark of APL, is associated with the outcome of APL
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treatment [28, 29]. However, according to some recent studies, PML/RARα is not
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necessary for the development of APL [6, 7]. In early myeloid cells, the PML/RARα
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fusion protein can be cleaved by NE, generating the NLS- negative PML and
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NLS-RARα. Both this paper and previous studies have shown that NLS-RARα can block the differentiation of AML cells [12, 13] (Figure 1A-E). T These results suggest
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that NLS-RARα plays a role in the pathogenic mechanism of APL. Further, we found
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that NLS-RARα mainly localized to the nucleus unlike RARα [10](Figure 2A-H). In
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previous studies, yeast two-hybrid assay results have shown that NLS-RARα could be associated with ubiquilin 1(UBQLN1) and JTV1 [30, 31], implying that the abnormal localization of NLS-RARα results in protein-protein interactions. Such interactions may change the original signaling pathway and accelerate the development of APL. Furthermore, the abnormal localization of the proteins is involved in many cancers [14, 32]. We postulate that the localization mechanism of NLS-RARα plays an important role in APL. Based on the predicted NLS and previous studies, NLS-RARα includes two NLSs, one each from PML and RARα (Supplementary Table 2). Previously, we speculated
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that the additional NLS increases the nuclear localization of NLS-RARα. In this study, we mutated the two NLSs and found that mutating only the NLS from the RARα portion could significantly decrease the nuclear localization of NLS-RARα. It demonstrated that the RARα portion plays a major role in NLS-RARα (Figure 3C-D). This is the first time that the NLS of the RARα portion have been shown to plays a major role in NLS-RARα localization. Further, the NLS-mutant NLS-RARα did not
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affect the differentiation of AML cells compared with wild NLS-RARα (Figure 4A-D).
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It verifies the nuclear accumulation of NLS-RARα restricted the differentiation once
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again.
The present study also uncovered the mechanism of NLS-RARα localization to the NLS-RARα possesses two
NLSs.
In
the
importin-α/β pathway,
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nucleus.
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cNLS-containing proteins assemble into a ternary complex with importin α and β [33].
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To determine whether NLS-RARα localization was mediated by importin α/β, immunoprecipitation and immunofluorescence analyses were performed. The results showed that NLS-RARα bound to importin-α1, which co- localized with importin-α/β (Figure 5C-D). Moreover, localization of NLS-RARα to the nucleus was markedly decreased in the presence of ivermectin and importazole, which specifically inhibit the importin-α/β pathway and importin-β pathway, respectively (Figure 6A-H). Taken together, these results showed that NLS-RARα is transported to the nucleus via the importin-α/β pathway.
RARα belongs to the nuclear receptor superfamily, which that recognizes retinoic
Journal Pre-proof acid (RA) as the ligand [34]. RARα regulates the transcription of genes containing the RAR response elements (RARE) [35]. In many types of cells, RARα is localized to the cytoplasm. In a previous study, RARα was localized to the cytoplasm under standard culture conditions, but in the presence of cytokines and RA, RARα gradually translocated to the nucleus [36]. However, the mechanism underlying the localization of RARα to the nucleus is still unclear. A study by Mezaki et al.
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demonstrated that endogenous RARα localized to the cytoplasm because the NLS of
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the endogenous RARα proteins was temporarily masked [25]. Further, a study by
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Huang showed that changes in the subcellular localization of RARα could be
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regulated by RA and protein kinase C [37]. Further, RARα is the phosphorylation substrate for the C-subunit of protein kinase A (PKA) and Ser-371 of RARα was
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predicted to be the phosphorylation site [38]. Studies have demonstrated that protein
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kinases regulate the localization of the target proteins containing NLS via the
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phosphorylation of protein kinase phosphorylation sites adjacent to the NLS [39, 40]. Hence, we speculated that RARα was normally located in the cytoplasm. However, the presence of ligands or phosphorylation, changed the conformation of RARα, resulting in exposure of the NLS, which allowed target proteins to enter the nucleus. NLS-RARα translocated to the nucleus based on the NLS of the RARα portion, and the transport of NLS-RARα may be analogous to that of RARα. Furthermore, we predicted that the putative phosphorylation sites were around the NLS in NLS-RARα using Netphos 3.1 Server (supplementary Figure 1). However, in APL patients, NLS-RARα localized to the nucleus. We speculate that NE cleaves PML/RARα,
Journal Pre-proof which results in conformational changes in NLS-RARα. This leads to exposure of the NLS and phosphorylation sites. However, further studies are needed to investigated this hypothesis. In conclusion, to the best of our knowledge, this is the first study to show the mechanism of nuclear localization of NLS-RARα, and to verify that nuclear accumulation of NLS-RARα decreases the differentiation of AML cells compared to
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its localization in the cytoplasm. These results suggest that NLS-RARα could be used
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as a biomarker in APL, and the transport pathway could be an alternative mechanism to
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modulate the pathogenic role of NLS-RARα in vitro.
Acknowledgments
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This work was funded by the National Natural Science Foundation of China (grant
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number 81772280) and the Chongqing Science and Technology Commission Fund
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（NO.cstc2018jscx-msybX0173）. Author contributions
Jiao Ye, Dongdong Liu and Juanjuan Yao conceived and designed the experiments; Jiao Ye, LiangZhong, Jiao Li, Li Xiong, Lihua Li, Pengqiang Zhong, performed the experiments; Zhen Yuan and Junmei Liu, analyzed the data; Jiao Ye wrote the paper. Declaration of competing Interest The authors declare that they have no conflict of interest. References [1]. de The H, Chen Z. Acute promyelocytic leukaemia: novel insights into the mechanisms of
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[34]. Yang N, Schule R, Mangelsdorf DJ, Evans RM. Characterization of DNA binding and
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[35]. Akmal KM, Dufour JM, Vo M, Higginson S, Kim KH. Ligand-dependent regulation of
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retinoic acid receptor alpha in rat testis: in vivo response to depletion and replet ion of
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GLI1.
Biochem
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2017;491(3):767-72.PMID:28735864
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Declaration of competing Interest
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The authors declare that they have no conflict of interest.
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Highlights 1.the nuclear localization of NLS-RARα inhibits 1,25D3- induced differentiation of HL-60 and U937 cells. 2.NLS of RARα portion mediates the nuclear localization of NLS-RARα. 3. importin-α/β participates in the nuclear transport of NLS-RARα via binding to
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NLS.